阅读说明：本技术 一种制备大黄鱼生物发酵饲料的方法 (Method for preparing large yellow croaker biological fermentation feed ) 是由 吴云通 黄贞胜 林鸿荣 于 2020-11-20 设计创作，主要内容包括：本发明公开了一种制备大黄鱼生物发酵饲料的方法,采用二种益生菌—枯草芽孢杆菌和植物乳杆菌作为发酵菌剂,豆粕作为发酵菌,并采用3立方固态发酵罐纯菌发酵取代传统的开放式发酵模式,便于控制发酵条件,缩短发酵周期；并有利于控制杂菌污染和有害产物生成,保证产品质量和安全,采用这种生产方式生产生物饲料有助于实现生产自动化,扩大生产规模,提高产品质量的稳定性。(The invention discloses a method for preparing a large yellow croaker biological fermentation feed, which adopts two probiotics, namely bacillus subtilis and lactobacillus plantarum, as fermentation inoculants, soybean meal as fermentation bacteria, and adopts a 3-cube solid-state fermentation tank pure-bacteria fermentation to replace the traditional open fermentation mode, thereby facilitating the control of fermentation conditions and shortening the fermentation period; the production method is favorable for realizing production automation, enlarging production scale and improving the stability of product quality.)

1. A method for preparing large yellow croaker biological fermentation feed is characterized in that: the method comprises the following steps:

s1, sequentially carrying out slant culture, activation culture, seed culture and fermentation culture on the lactobacillus plantarum to obtain lactobacillus plantarum viable bacteria;

s2, sequentially carrying out activation culture, seed culture and fermentation culture on the bacillus subtilis to obtain live bacillus subtilis, wherein the culture method of the activation culture is a plate marking method;

s3, fermenting bean pulp: inoculating soybean meal with initial water content of 50% into a solid fermentation culture medium, and respectively sequentially inoculating lactobacillus plantarum viable bacteria and bacillus subtilis viable bacteria for fermentation culture to obtain fermented soybean meal;

s4, adding the fermented soybean meal into the large yellow croaker feed to obtain the fermented feed.

2. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: the specific culture methods of slant culture, activation culture, seed culture and fermentation culture of lactobacillus plantarum in the step S1 are respectively as follows:

slant culture of lactobacillus plantarum: culturing the inoculation slant of the lactobacillus plantarum in a constant temperature incubator at 37 ℃ for 36h, then placing the culture in a refrigerator at 4 ℃ for preservation, and carrying out passage once every two weeks to obtain slant culture thalli;

activating and culturing lactobacillus plantarum: inoculating 2-3 rings of the slant culture thalli into a 250mL triangular flask filled with 100mL liquid seed culture medium, and culturing at 37 ℃ for 24h at 50r/min to obtain activated lactobacillus plantarum;

culturing lactobacillus plantarum seeds: inoculating the activated lactobacillus plantarum into a 250mL triangular flask, and filling 100mL of seed culture medium into the 250mL triangular flask, wherein the inoculation amount is 2%, and culturing is carried out at 37 ℃ and 50r/min for 14h to obtain seed lactobacillus plantarum;

and (3) fermenting and culturing lactobacillus plantarum: inoculating the seed lactobacillus plantarum into a 250mL triangular flask, filling 100mL fermentation medium into the 250mL triangular flask, wherein the inoculation amount is 3%, and culturing at 37 ℃ and 50r/min for 14h to obtain lactobacillus plantarum viable bacteria.

3. The method for preparing the large yellow croaker biofermented feed according to claim 2, wherein: the liquid seed culture medium for lactobacillus plantarum activation culture, the seed culture medium for lactobacillus plantarum seed culture and the fermentation culture medium for lactobacillus plantarum fermentation culture all comprise the following components: 20g/L of glucose, 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast extract powder, 2g/L of diammonium hydrogen citrate, 3COONa & 3H2O 5g/L, 42K 2HPO 42 g/L, 4 & 7H2O 0.58.58 g/L, MnSO4 & H2O 0.25.25 g/L and 801 mL of Tween, wherein the pH value of each culture medium is 6.4.

4. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: the specific culture methods of the activation culture, the seed culture and the fermentation culture of the bacillus subtilis in the step S2 are respectively as follows:

activating and culturing bacillus subtilis: inoculating a bacillus subtilis strain on a nutrient agar culture medium of a beef extract peptone solid plate by adopting a plate-scribing method, culturing for 24 hours in a constant-temperature incubator at 37 ℃, placing in a refrigerator at 4 ℃, and carrying out passage once every two weeks to obtain activated bacillus subtilis;

b, culturing the bacillus subtilis seeds: selecting the activated single colony to be cultured in a 250mL triangular flask filled with 100mL liquid seed culture medium for 20h at 37 ℃ at 200r/min to obtain seed bacillus subtilis;

b, fermentation culture of the bacillus subtilis: inoculating the seed bacillus subtilis into a 250mL triangular flask, filling 100mL of fermentation medium into the 250mL triangular flask, adjusting the initial pH to 7.0 and the inoculation amount to be 2%, and culturing at 37 ℃ and 200r/min for 20h to obtain the bacillus subtilis viable bacteria.

5. The method for preparing the large yellow croaker biofermented feed according to claim 4, wherein: the nutrient agar culture medium in the activation culture of the bacillus subtilis comprises the following components: 3g/L of beef extract, 10g/L of bacteriological peptone, 5g/L of NaCl and 20g/L of agar, wherein the pH value of a nutrient agar culture medium is 7.0;

the seed culture medium in the bacillus subtilis seed culture comprises the following components: 3g/L of soybean peptone, 10g/L of bacteriological peptone, 5g/L of NaCl, and the pH value of a seed culture medium is 7.0;

the fermentation medium in the fermentation culture of the bacillus subtilis comprises the following components: 15g/L of soluble starch, 7.5g/L of yeast powder, 1g/L of MgSO4.7H2O 2 and KCl, and the pH value of the fermentation medium is 7.0.

6. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: the solid state fermentation medium in the step S3 comprises the following components: 50g of soybean meal and 50mL of distilled water, and the pH value of the solid fermentation medium is natural.

7. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: the inoculation amounts of the live lactobacillus plantarum bacteria and the live bacillus subtilis bacteria in the step S3 are respectively as follows: the inoculation amount of the bacillus subtilis is 8 percent, and the inoculation ratio of the bacillus subtilis to the lactobacillus plantarum is 1: 5.

8. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: in the step S3, the inoculation sequence and the fermentation time of the live lactobacillus plantarum and the live bacillus subtilis are respectively as follows: inoculating Bacillus subtilis for 2 days, inoculating Lactobacillus plantarum, and fermenting for 5 days.

9. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: the fermentation temperature of the soybean meal fermentation in the step S3 is 37 ℃.

10. The method for preparing the large yellow croaker biofermented feed according to claim 1, wherein: in the step S4, 22 percent of fermented soybean meal with the protein content of 50 percent is added to replace 30 percent of fish meal, and the mixture ratio of the raw materials of the replaced fermented feed is as follows: 0.1 percent of fish meal with protein content of 67 percent, 0.075 percent of fish meal with protein content of 65 percent, 0.08 percent of chicken meal with protein content of 67 percent, 0.05 percent of corn protein powder with protein content of 60 percent, 0.1 percent of soybean meal with protein content of 43 percent, 0.22 percent of fermented soybean meal with protein content of 50 percent, 0.2 percent of soybean oil, 0.5 percent of fish oil, 0.15 percent of flour with protein content of 13 percent, 0.025 percent of cassava raw powder and 0.8 percent of premix.

Technical Field

The invention relates to the technical field of biological feed preparation, in particular to a method for preparing a large yellow croaker biological fermentation feed.

Background

China is the only world with the aquaculture yield exceeding the fishing yield, the aquaculture yield is at the top of the world, Fujian provinces are building the core region of 21 st century maritime silk roads, and mariculture is one of the key points. With the development of aquaculture industry, the intensification degree is constantly improved, and the use of artifical compound feed in a large number in the breeding process has produced some problems that await solution urgently, if: the feed has the advantages that the digestibility is reduced, the feed coefficient is increased, physical and chemical indexes and biological factors in the water environment are changed due to excessive excrement and residual bait in the aquaculture water, the self-purification capacity of the water is reduced, the water eutrophication or water quality is deteriorated, a large number of pathogens are bred, new pathogens continuously appear, and the problems of increasing the occurrence frequency of diseases of aquaculture animals, slow growth and the like are caused. In order to prevent and treat diseases, chemical medicines such as antibiotics and the like are applied to aquaculture in a large quantity, and the use of the antibiotics brings about the problems of bacterial drug resistance, residues, water body pollution and the like, so that the healthy and sustainable development of the aquaculture industry is restricted. Therefore, the development and application of the high-efficiency environment-friendly biological fermentation feed which has no pollution, no residue, growth promotion and immunity enhancement have important practical significance for the aquaculture industry in the new period. Based on the method, the invention designs a method for preparing the large yellow croaker biological fermentation feed.

Disclosure of Invention

The invention aims to provide a method for preparing a large yellow croaker biological fermentation feed, which aims to solve the technical problems.

In order to achieve the purpose, the invention provides the following technical scheme: a method for preparing a large yellow croaker biological fermentation feed comprises the following steps:

s1, sequentially carrying out slant culture, activation culture, seed culture and fermentation culture on the lactobacillus plantarum to obtain lactobacillus plantarum viable bacteria;

s2, sequentially carrying out activation culture, seed culture and fermentation culture on the bacillus subtilis to obtain live bacillus subtilis, wherein the culture method of the activation culture is a plate marking method;

s3, fermenting bean pulp: inoculating soybean meal with initial water content of 50% into a solid fermentation culture medium, and respectively sequentially inoculating lactobacillus plantarum viable bacteria and bacillus subtilis viable bacteria for fermentation culture to obtain fermented soybean meal;

s4, adding the fermented soybean meal into the large yellow croaker feed to obtain the fermented feed.

Preferably, the specific culture methods of the slant culture, the activation culture, the seed culture and the fermentation culture of lactobacillus plantarum in step S1 are respectively as follows:

slant culture of lactobacillus plantarum: culturing the inoculation slant of the lactobacillus plantarum in a constant temperature incubator at 37 ℃ for 36h, then placing the culture in a refrigerator at 4 ℃ for preservation, and carrying out passage once every two weeks to obtain slant culture thalli;

activating and culturing lactobacillus plantarum: inoculating 2-3 rings of the slant culture thalli into a 250mL triangular flask filled with 100mL liquid seed culture medium, and culturing at 37 ℃ for 24h at 50r/min to obtain activated lactobacillus plantarum;

culturing lactobacillus plantarum seeds: inoculating the activated lactobacillus plantarum into a 250mL triangular flask, and filling 100mL of seed culture medium into the 250mL triangular flask, wherein the inoculation amount is 2%, and culturing is carried out at 37 ℃ and 50r/min for 14h to obtain seed lactobacillus plantarum;

and (3) fermenting and culturing lactobacillus plantarum: inoculating the seed lactobacillus plantarum into a 250mL triangular flask, filling 100mL fermentation medium into the 250mL triangular flask, wherein the inoculation amount is 3%, and culturing at 37 ℃ and 50r/min for 14h to obtain lactobacillus plantarum viable bacteria.

Preferably, the liquid seed culture medium for lactobacillus plantarum activation culture, the seed culture medium for lactobacillus plantarum seed culture and the fermentation culture medium for lactobacillus plantarum fermentation culture all comprise the following components: 20g/L of glucose, 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast extract powder, 2g/L of diammonium hydrogen citrate, 3COONa & 3H2O 5g/L, 42K 2HPO 42 g/L, 4 & 7H2O 0.58.58 g/L, MnSO4 & H2O0.25g/L and 801 mL of Tween, wherein the pH value of each culture medium is 6.4.

Preferably, the specific culture methods of the activation culture, the seed culture and the fermentation culture of the bacillus subtilis in the step S2 are respectively as follows:

activating and culturing bacillus subtilis: inoculating a bacillus subtilis strain on a nutrient agar culture medium of a beef extract peptone solid plate by adopting a plate-scribing method, culturing for 24 hours in a constant-temperature incubator at 37 ℃, placing in a refrigerator at 4 ℃, and carrying out passage once every two weeks to obtain activated bacillus subtilis;

b, culturing the bacillus subtilis seeds: selecting the activated single colony to be cultured in a 250mL triangular flask filled with 100mL liquid seed culture medium for 20h at 37 ℃ at 200r/min to obtain seed bacillus subtilis;

b, fermentation culture of the bacillus subtilis: inoculating the seed bacillus subtilis into a 250mL triangular flask, filling 100mL of fermentation medium into the 250mL triangular flask, adjusting the initial pH to 7.0 and the inoculation amount to be 2%, and culturing at 37 ℃ and 200r/min for 20h to obtain the bacillus subtilis viable bacteria.

Preferably, the nutrient agar culture medium in the activation culture of the bacillus subtilis comprises the following components: 3g/L of beef extract, 10g/L of bacteriological peptone, 5g/L of NaCl and 20g/L of agar, wherein the pH value of a nutrient agar culture medium is 7.0;

the seed culture medium in the bacillus subtilis seed culture comprises the following components: 3g/L of soybean peptone, 10g/L of bacteriological peptone, 5g/L of NaCl, and the pH value of a seed culture medium is 7.0;

the fermentation medium in the fermentation culture of the bacillus subtilis comprises the following components: 15g/L of soluble starch, 7.5g/L of yeast powder, 1g/L of MgSO4.7H2O 2 and KCl, and the pH value of the fermentation medium is 7.0.

Preferably, the solid state fermentation medium in step S3 comprises: 50g of soybean meal and 50mL of distilled water.

Preferably, the inoculation amounts of the live lactobacillus plantarum and the live bacillus subtilis in step S3 are respectively as follows: the inoculation amount of the bacillus subtilis is 8 percent, and the inoculation ratio of the bacillus subtilis to the lactobacillus plantarum is 1: 5.

Preferably, in the step S3, the inoculation sequence and fermentation time of the live lactobacillus plantarum and the live bacillus subtilis are respectively as follows: inoculating Bacillus subtilis for 2 days, inoculating Lactobacillus plantarum, and fermenting for 5 days.

Preferably, the fermentation temperature of the soybean meal fermentation in the step S3 is 37 ℃.

Preferably, in the step S4, 22% of fermented soybean meal with a protein content of 50% is added to replace 30% of fish meal, and the substituted fermented feed comprises the following raw materials in parts by weight: 0.1 percent of fish meal with protein content of 67 percent, 0.075 percent of fish meal with protein content of 65 percent, 0.08 percent of chicken meal with protein content of 67 percent, 0.05 percent of corn protein powder with protein content of 60 percent, 0.1 percent of soybean meal with protein content of 43 percent, 0.22 percent of fermented soybean meal with protein content of 50 percent, 0.2 percent of soybean oil, 0.5 percent of fish oil, 0.15 percent of flour with protein content of 13 percent, 0.025 percent of cassava raw powder and 0.8 percent of premix.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, two probiotics, namely bacillus subtilis and lactobacillus plantarum, are used as fermentation inoculants, soybean meal is used as fermentation bacteria, and pure bacteria fermentation of a 3-cube solid-state fermentation tank is adopted to replace a traditional open fermentation mode, so that the fermentation conditions are convenient to control, and the fermentation period is shortened; the production method is favorable for realizing production automation, enlarging production scale and improving the stability of product quality.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a manufacturing method of the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, the present invention provides a technical solution: in particular to a method for preparing large yellow croaker biological fermentation feed, which comprises the following steps:

s1, sequentially carrying out slant culture, activation culture, seed culture and fermentation culture on the lactobacillus plantarum to obtain lactobacillus plantarum viable bacteria;

s2, sequentially carrying out activation culture, seed culture and fermentation culture on the bacillus subtilis to obtain live bacillus subtilis, wherein the culture method of the activation culture is a plate marking method;

s3, fermenting bean pulp: inoculating soybean meal with initial water content of 50% into a solid fermentation culture medium, and respectively sequentially inoculating lactobacillus plantarum viable bacteria and bacillus subtilis viable bacteria for fermentation culture to obtain fermented soybean meal;

s4, adding the fermented soybean meal into the large yellow croaker feed to obtain the fermented feed.

Specifically, the specific culture methods of slant culture, activation culture, seed culture and fermentation culture of lactobacillus plantarum in step S1 are as follows:

slant culture of lactobacillus plantarum: culturing the inoculation slant of the lactobacillus plantarum in a constant temperature incubator at 37 ℃ for 36h, then placing the culture in a refrigerator at 4 ℃ for preservation, and carrying out passage once every two weeks to obtain slant culture thalli;

activating and culturing lactobacillus plantarum: inoculating 2-3 rings of the slant culture thalli into a 250mL triangular flask filled with 100mL liquid seed culture medium, and culturing at 37 ℃ for 24h at 50r/min to obtain activated lactobacillus plantarum;

culturing lactobacillus plantarum seeds: inoculating the activated lactobacillus plantarum into a 250mL triangular flask, and filling 100mL of seed culture medium into the 250mL triangular flask, wherein the inoculation amount is 2%, and culturing is carried out at 37 ℃ and 50r/min for 14h to obtain seed lactobacillus plantarum;

and (3) fermenting and culturing lactobacillus plantarum: inoculating the seed lactobacillus plantarum into a 250mL triangular flask, filling 100mL fermentation medium into the 250mL triangular flask, wherein the inoculation amount is 3%, and culturing at 37 ℃ and 50r/min for 14h to obtain lactobacillus plantarum viable bacteria.

Specifically, the liquid seed culture medium for lactobacillus plantarum activation culture, the seed culture medium for lactobacillus plantarum seed culture and the fermentation culture medium for lactobacillus plantarum fermentation culture all comprise the following components: 20g/L of glucose, 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast extract powder, 2g/L of diammonium hydrogen citrate, 3COONa & 3H2O 5g/L, 42K 2HPO 42 g/L, 4 & 7H2O 0.58.58 g/L, MnSO4 & H2O0.25g/L and 801 mL of Tween, wherein the pH value of each culture medium is 6.4.

Specifically, the specific culture methods of the activation culture, the seed culture and the fermentation culture of the bacillus subtilis in the step S2 are respectively as follows:

activating and culturing bacillus subtilis: inoculating a bacillus subtilis strain on a nutrient agar culture medium of a beef extract peptone solid plate by adopting a plate-scribing method, culturing for 24 hours in a constant-temperature incubator at 37 ℃, placing in a refrigerator at 4 ℃, and carrying out passage once every two weeks to obtain activated bacillus subtilis;

b, culturing the bacillus subtilis seeds: selecting the activated single colony to be cultured in a 250mL triangular flask filled with 100mL liquid seed culture medium for 20h at 37 ℃ at 200r/min to obtain seed bacillus subtilis;

b, fermentation culture of the bacillus subtilis: inoculating the seed bacillus subtilis into a 250mL triangular flask, filling 100mL of fermentation medium into the 250mL triangular flask, adjusting the initial pH to 7.0 and the inoculation amount to be 2%, and culturing at 37 ℃ and 200r/min for 20h to obtain the bacillus subtilis viable bacteria.

Specifically, the nutrient agar culture medium in the activation culture of the bacillus subtilis comprises the following components: 3g/L of beef extract, 10g/L of bacteriological peptone, 5g/L of NaCl and 20g/L of agar, wherein the pH value of a nutrient agar culture medium is 7.0;

the seed culture medium in the bacillus subtilis seed culture comprises the following components: 3g/L of soybean peptone, 10g/L of bacteriological peptone, 5g/L of NaCl, and the pH value of a seed culture medium is 7.0;

the fermentation medium in the fermentation culture of the bacillus subtilis comprises the following components: 15g/L of soluble starch, 7.5g/L of yeast powder, 1g/L of MgSO4.7H2O 2 and KCl, and the pH value of the fermentation medium is 7.0.

Specifically, the solid state fermentation medium in step S3 includes the following components: 50g of soybean meal and 50mL of distilled water.

Specifically, the inoculation amounts of the live lactobacillus plantarum bacteria and the live bacillus subtilis bacteria in the step S3 are respectively as follows: the inoculation amount of the bacillus subtilis is 8 percent, and the inoculation ratio of the bacillus subtilis to the lactobacillus plantarum is 1: 5.

Specifically, in the step S3, the inoculation sequence and fermentation time of the live lactobacillus plantarum and the live bacillus subtilis are respectively as follows: inoculating Bacillus subtilis for 2 days, inoculating Lactobacillus plantarum, and fermenting for 5 days.

Specifically, the fermentation temperature of the soybean meal fermentation in the step S3 is 37 ℃.

Specifically, in the step S4, 22% of fermented soybean meal with a protein content of 50% is added to replace 30% of fish meal, and the substituted fermented feed comprises the following raw materials in parts by weight: 0.1 percent of fish meal with protein content of 67 percent, 0.075 percent of fish meal with protein content of 65 percent, 0.08 percent of chicken meal with protein content of 67 percent, 0.05 percent of corn protein powder with protein content of 60 percent, 0.1 percent of soybean meal with protein content of 43 percent, 0.22 percent of fermented soybean meal with protein content of 50 percent, 0.2 percent of soybean oil, 0.5 percent of fish oil, 0.15 percent of flour with protein content of 13 percent, 0.025 percent of cassava raw powder and 0.8 percent of premix.

The first embodiment is as follows:

s1, sequentially carrying out slant culture, activation culture, seed culture and fermentation culture on the lactobacillus plantarum to obtain lactobacillus plantarum viable bacteria;

the specific culture methods of slant culture, activation culture, seed culture and fermentation culture of lactobacillus plantarum in step S1 are respectively as follows:

slant culture of lactobacillus plantarum: culturing the inoculation slant of the lactobacillus plantarum in a constant temperature incubator at 37 ℃ for 36h, then placing the culture in a refrigerator at 4 ℃ for preservation, and carrying out passage once every two weeks to obtain slant culture thalli;

activating and culturing lactobacillus plantarum: inoculating 2-3 rings of the slant culture thalli into a 250mL triangular flask filled with 100mL liquid seed culture medium, and culturing at 37 ℃ for 24h at 50r/min to obtain activated lactobacillus plantarum;

culturing lactobacillus plantarum seeds: inoculating the activated lactobacillus plantarum into a 250mL triangular flask, and filling 100mL of seed culture medium into the 250mL triangular flask, wherein the inoculation amount is 2%, and culturing is carried out at 37 ℃ and 50r/min for 14h to obtain seed lactobacillus plantarum;

and (3) fermenting and culturing lactobacillus plantarum: inoculating the seed lactobacillus plantarum into a 250mL triangular flask, and filling 100mL fermentation medium into the 250mL triangular flask, wherein the inoculation amount is 3%, and culturing is carried out at 37 ℃ and 50r/min for 14h to obtain lactobacillus plantarum viable bacteria;

the liquid seed culture medium for lactobacillus plantarum activation culture, the seed culture medium for lactobacillus plantarum seed culture and the fermentation culture medium for lactobacillus plantarum fermentation culture all comprise the following components: 20g/L of glucose, 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast extract powder, 2g/L of diammonium hydrogen citrate, 3 COONa.3H.2 2O 5g/L, K2HPO 42 g/L, 0.58g/L of MgSO4.7 H.2 2O 0.58, 0.25g/L of MnSO 4.H.2 2O0.25 and 801 mL of Tween, wherein the pH value of each culture medium is 6.4;

s2, sequentially carrying out activation culture, seed culture and fermentation culture on the bacillus subtilis to obtain live bacillus subtilis, wherein the culture method of the activation culture is a plate marking method;

activating and culturing bacillus subtilis: inoculating bacillus subtilis strain on a nutrient agar culture medium of a beef extract peptone solid plate by adopting a plate-scribing method, culturing for 24 hours in a constant-temperature incubator at 37 ℃, placing in a refrigerator at 4 ℃, and carrying out passage once every two weeks to obtain activated bacillus subtilis, wherein the nutrient agar culture medium in the activated culture of the bacillus subtilis comprises the following components: 3g/L of beef extract, 10g/L of bacteriological peptone, 5g/L of NaCl and 20g/L of agar, wherein the pH value of a nutrient agar culture medium is 7.0;

b, culturing the bacillus subtilis seeds: and (3) selecting the activated single colony to be cultured in a 250mL triangular flask filled with 100mL liquid seed culture medium at 37 ℃ at 200r/min for 20h to obtain the seed bacillus subtilis, wherein the seed culture medium in the bacillus subtilis seed culture comprises the following components: 3g/L of soybean peptone, 10g/L of bacteriological peptone, 5g/L of NaCl, and the pH value of a seed culture medium is 7.0;

b, fermentation culture of the bacillus subtilis: inoculating the seed bacillus subtilis into a 250mL triangular flask, filling 100mL of fermentation medium into the 250mL triangular flask, adjusting the initial pH to 7.0 and the inoculation amount to be 2%, culturing at 37 ℃ and 200r/min for 20h to obtain the bacillus subtilis viable bacteria, wherein the fermentation medium in the bacillus subtilis fermentation culture comprises the following components: 15g/L of soluble starch, 7.5g/L of yeast powder, 1g/L of MgSO4.7H2O 2 and KCl, and the pH value of a fermentation medium is 7.0;

s3, fermenting bean pulp: inoculating soybean meal with initial water content of 50% into a solid fermentation culture medium, respectively and sequentially inoculating lactobacillus plantarum viable bacteria and bacillus subtilis viable bacteria for fermentation culture, wherein the inoculation amount of the bacillus subtilis is 8%, the inoculation ratio of the bacillus subtilis to the lactobacillus plantarum is 1:5, then inoculating the bacillus subtilis for 2 days, then inoculating the lactobacillus plantarum, and fermenting for 5 days to obtain fermented soybean meal, wherein the solid fermentation culture medium comprises the following components: 50g of soybean meal and 50mL of distilled water, wherein the fermentation temperature is 37 ℃;

s4, in the step S4, 22 percent of fermented soybean meal with the protein content of 50 percent is added to replace 30 percent of fish meal to obtain fermented feed, and the mixture ratio of the raw materials of the replaced fermented feed is as follows: 0.1 part of fish meal with the protein content of 67 percent, 0.075 part of fish meal with the protein content of 65 percent, 0.08 part of chicken meal with the protein content of 67 percent, 0.05 part of corn protein powder with the protein content of 60 percent, 0.1 part of bean pulp with the protein content of 43 percent, 0.22 part of fermented bean pulp with the protein content of 50 percent, 0.2 part of soybean oil, 0.5 part of fish oil, 0.15 part of flour with the protein content of 13 percent, 0.025 part of cassava raw powder and 0.8 part of premix;

example two:

the difference from the first embodiment is that: s4, adding 41.5% of fermented soybean meal with protein content of 50% to replace 50% of fish meal to obtain a fermented feed, wherein the substituted fermented feed comprises the following raw materials in parts by weight: 0.1 percent of fish meal with protein content of 67 percent, 0.025 percent of fish meal with protein content of 65 percent, 0.08 percent of chicken meal with protein content of 67 percent, 0.05 percent of corn protein powder with protein content of 60 percent, 0.415 percent of fermented soybean meal with protein content of 50 percent, 0.25 percent of soybean oil, 0.5 percent of fish oil, 0.15 percent of flour with protein content of 13 percent, 0.025 percent of cassava raw powder and 0.8 percent of premix.

Comparative example one:

the difference from the first embodiment is that: the raw yellow croaker feed is adopted, and the raw material ratio of the raw yellow croaker feed is as follows: 0.1 percent of fish meal with protein content of 67 percent, 0.1 percent of fish meal with protein content of 65 percent, 0.05 percent of fish meal with protein content of 60 percent, 0.08 percent of chicken meal with protein content of 67 percent, 0.05 percent of corn protein powder with protein content of 60 percent, 0.3 percent of soybean meal with protein content of 43 percent, 0.15 percent of soybean oil, 0.5 percent of fish oil, 0.15 percent of flour with protein content of 13 percent, 0.025 percent of cassava raw powder and 0.8 percent of premix.

The large yellow croaker feeds in the comparative example (group a), the first example (group B) and the second example (group C) were subjected to an application experiment of the culture effect, which was conducted in a cement pond in a greenhouse having a steam heating facility, and the seawater salinity was 22; p H value is 8.2; the experimental water temperature is controlled to be (22 +/-1) ° C; the water body is continuously aerated, the value of the aerated water body p H is 7.9-8.0, and the dissolved oxygen is 5.17-7.13 mg/L during the experiment; the ammonia nitrogen is less than 0.01 mg/L. During the test, water 1/5 was changed every day, and feces and residual bait at the bottom of the tank were sucked off. The experimental fish is fed twice in morning and evening every day, the feeding amount is flexibly controlled according to the weather and the ingestion condition of the fish, and the experimental fish is generally finished when the fish school floats upwards to ingest and sinks and then a small amount of the experimental fish is fed; the experiment site is applied to an XX farm, six cement ponds of 4 meters by 4 meters are respectively selected for carrying out the experiment for 60 days, and the following data are obtained:

1. growth performance (as shown in Table 1)

TABLE 1 Pseudosciaena crocea biological fermentation feedstuff feeding experimental result

Group of	A	B	C
				Average tail initial weight/g	20.6	20.6	20.6
Stocking mantissa	2500	2500	2500
				Total amount of fish placed and cultured/kg	51.50	51.50	51.50
Total amount of harvested fish/kg	62.951	85.137	71.33
				Harvest mantissa	1955	2405	2155
Average tail end weight/g	32.2	35.4	33.1
				Total weight gain/kg of fish	11.451	33.637	19.83
Feed amount/kg	12.138	32.291	20.028
				Survival rate/%	78.2	90.6	86.2
Average tail weight gain/g	11.6	14.8	12.5
				Weight gain%	56.3	71.8	60.7
Coefficient of bait	1.06	0.96	1.01
				Feed efficiency/%)	94.3	104.2	99

As can be seen from table 1, the experimental results show that: the feed efficiency of the group B is improved by 10.5 percent compared with the group A, and the feed efficiency of the group C is improved by 4.98 percent compared with the group A. The survival rate of the group B is improved by 15.9/%, and the survival rate of the group C is improved by 10.2% compared with the group A.

2. Non-specific immunity (as shown in Table 2)

TABLE 2 influence of fermented feed on nonspecific immunity of large yellow croaker

Group of	A	B	C
				Acid phosphatase (μmol/min/mL)	0.24	0.43	0.36
Alkaline phosphatase (μmol/min/mL)	0.28	0.53	0.35
				Catalase (μmol/min/mL)	24.85	17.11	12.79
Superoxide dismutase (U/mL)	3.66	11.48	7.12
				Lysozyme (mu g/mL)	634.26	953.59	755.78

As can be seen from Table 2, the alkaline phosphatase and the acid phosphatase in the experimental group have higher enzyme activities than the control group, and both the groups B > C > A. The catalase activity of the experimental group is higher than that of the control group, which shows that the catalase activity can be improved through laminarin. The catalase activity of each experimental group is lower than that of a control group, which indicates that the biological fermentation feed for the large yellow croakers cannot improve the catalase activity. Superoxide dismutase (SOD) can remove excessive free radicals in the organism, is an important index for reflecting the oxidation resistance of the organism, and results show that the SOD activity of an experimental group is obviously higher than that of a control group, and the activity of a group B is greater than that of a group C and is greater than that of a group A. Lysozyme exists in neutrophils, monocytes and macrophages, is one of important components of immune defense systems of vertebrates and non-vertebrates, and can hydrolyze peptidoglycan in cell walls of gram-positive bacteria to crack the cells, thereby killing pathogenic microorganisms. The lysozyme activities of the experimental groups were all higher than those of the control group in this experiment, and group B > group C > group a.

3. Digestive enzymes (as shown in Table 3)

TABLE 3 influence of fermented feed on digestive enzyme of large yellow croaker

Group of	A	B	C
				Lipase (U/mL)	0	6.48	4.15
Protease (U/g)	48.65	92.84	75.12
				Amylase (U/dl)	320.72	531.76	408.08

As can be seen from table 3, in the lipase enzyme activity assay, the control group a had no enzyme activity, whereas the experimental groups had enzyme activity, and group B > group C. The protease and amylase in the experimental group are higher than those in the control group, and the protease and the amylase in the experimental group are both group B > group C > group A.

And (4) conclusion: to sum up the results of the experiments, the group B (fermented soybean meal with 50% of addition ratio of 22% instead of 30% of fish meal) has the best breeding effect.

Although embodiments of the present invention have been shown and described, it would be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the claims and their equivalents.